The long-term goal of this project is to better understand Borrelia burgdorferi (Bb) motility as it relates to pathogenesis. The central hypothesis is that Bb and other spirochete species contain a unique cross-linking of the flagellar hook protein FlgE that is essential for optimal motility. The two aims below investigate the identification and formation of the cross-links. Understanding FlgE cross-link synthesis and structure and can potentially lead to the development of novel drugs that inhibit this cross-linking. These drugs could potentially be used to treat Lyme borrelosis and other spirochetal diseases. Specific aim 1. Based on published and preliminary data, we hypothesize that the Bb FlgE flagellar hook proteins are covalently cross-linked to one another. To test this hypothesis, hook- basal-body complexes will be purified from growing cells, and recombinant Bb FlgE (rFlgE) will be synthesized in Escherichia coli and purified as described. The hook basal body complexes and rFlgE will be digested with agents such as trypsin, and the products will be separated and analyzed by mass spectrometry. Differences in the peptide patterns will indicate which peptides are cross-linked. The specific amino acids and nature of the bond involved in the cross-linking will be determined using peptide mapping and tandem mass spectrometry. Specific aim 2. We hypothesize that site-directed mutations that alter specific residues in Bb flgE result in the inability of the hook protein FlgE to be cross-linked. Cells bearing such mutations are also hypothesized to have altered motility. To test these hypotheses, two approaches will be used to construct flgE mutants. First, specific mutations in the plasmid flgE/pBSV2 containing cloned Bb flgE will be constructed. This mutant has been shown to complement the flgE non-motile insertion mutant SC-E1. The mutagenized plasmids will be electroporated into SC-E1 and analyzed as described below Second, specific mutations in the chromosomally encoded flgE will be constructed using a recently developed counter-selection technique. Mutants generated by both methods will be tested for synthesis of FlgE, FlgE cross-linking, flagellar hook production and length, and motilty. The methods employed include western blotting for FlgE synthesis and cross-linking, transmission electron microscopy for flagellar hook synthesis and length determination, and swarm plate assays and computerized cell tracking to assess altered motility; the investigators have extensive experience using all these techniques. The results obtained should allow for the identification of the amino acids involved in FlgE cross-linking and the effects of this cross-linking on Bb motility. PUBLIC HEALTH RELEVANCE: The ability of bacteria to move is important for many of them to cause disease; many swim by rotating their flagella, one component of which are the flexible 'hooks'. Spirochetes cause many diseases world-wide, and they appear to exhibit a unique cross-linking of hook proteins. By understanding this cross-linking, novel drugs can be developed to target this cross-linking and combat spirochete-caused diseases.